Mouse hepatitis virus provides an instructive model to investigate the host and virus dependent mechanisms which result in demyelination. Neurotropic MHV-JHM infection typically exhibits an acute phase characterized by virus infection and replication to high titers in CNS neurons and glia followed closely by T-cell dependent control of infection and disappearance of infectious virus by 11-15 days. Viral RNA persists in the white matter in astrocytes and oligodendrocytes for extended periods of time. Coincident with the appearance of virus in white matter tracts is the appearance of an acute demyelinating pathology characterized by mononuclear infiltration and white matter destruction followed by a chronic stage marked by active macrophage phagocytosis of myelin debris and remyelination. Viral RNA remains detectable for extended periods and is likely to provide the primary stimulus for white matter destruction. It has been established that the spike protein, S, plays a crucial role in infection by defining the receptor recognition and binding by the virus, and providing the fusion function required by the virus for cell entry and for cell to cell spread. In the next project period we will focus our efforts on modelling the functional domains of S with the goal of addressing the mechanisms by which MHV-JHM S protein function to control viral tropism, fusion and entry. Two related specific aims proposed for the next project period are: 1. Modeling conformational changes associated with fusion activation in the MHV-JHM and OBLV60 spikes by cryoelectron microscopy, peptide modeling and fusion assays. 2. Analysis of persistent viral RNA in vivo to determine whether mutations in the genome occur which affect asembly or fusion.